The concept of gene therapy first arose during the 1960s and early 1970s. As it became clear that active genes may be transferred into mammalian cells, the idea that diseases may be treated at the genetic level started to develop. In many hereditary diseases the patient suffers from low or altogether absent production of a critical protein. The rationale behind gene therapy approach is that the missing protein may be supplied by the appropriate gene, encoding the specific protein, introduced into the patient's cells. This rationale may be applied to any protein of therapeutic value, for example the production of tumor suppressors in the treatment of cancer. Furthermore, this approach is also applicable to the production of regulatory macromolecules such nucleic acids which would bind to a biologically active protein or block its expression.
There are two basic strategies, generally ex vivo and in vivo gene therapy. Ex vivo gene therapy may be described as genetically modifying cells obtained from a patient, or from a different source, and using the modified cells to treat a pathological condition which will be improved by the long-term or constant delivery of the therapeutic product produced by the modified cells. Treatment includes also the re-introduction, where applicable, e.g. in the case of erythroid cells, of the modified cells, obtained from either the patient or from a different source, into the patient. In vivo gene therapy, on the other hand, refers to direct in vivo delivery of the therapeutic gene into the appropriate tissue of the patient. In vivo delivery may be achieved by a variety of techniques, such as intravenous delivery, direct injection into muscles, inhalation into the lung, etc.
With respect to the specific case of treating .beta.-thalassemia, i.e. gene therapy with the normal .beta.-globin gene, a number of developments have been achieved. The molecular defects of .beta.-thalassemia have been well characterized and seem amenable to genetic correction. In .beta.-thalassemia patients, deficient or absent .beta.-globin gene synthesis causes the production of poorly hemoglobinized, defective erythrocytes resulting in severe anemia. Effective gene therapy requires safe, efficient, and stable transfer of globin genes into human hemopoietic stem cells and subsequent high-level gene expression in maturing erythroid cells. Prior to clinical trials, it was necessary to develop experimental laboratory and animal models. Such models would enable the detailed study of the target cells and the regulation of the inserted gene.
One of the major problems in developing a model for gene therapy of .beta.-thalassemia is the difficulty in introducing the cloned plasmid DNA into hemopoietic cells. Several procedures have been developed to circumvent this difficulty. However, none has been successful in achieving gene delivery and expression. For example, attempts have been made to insert the .beta.-globin gene into retrovirus-derived vectors and subsequently to infect multi-potential hemopoietic progenitors of murine erythroid cells [see Karlsson, S., et al., (1988) Proc. Natl. Acad. Sci. USA 85:6062-6066]. While .beta.-globin expression was obtained in the transduced animals and cells, this system using retrovirus-derived vectors is undesirable for several reasons. Use of retrovirus-derived vectors would be inefficient with human cells and incompatible with non-dividing cells [Miller, D. G., et al., Mol. Cell. Biol. (1990) 10(8):4239-4242] and also causes massive rearrangements when LCR elements are included [Chang, J. C., et al., (1992) Proc. Natl. Acad. Sci. 89:3107-3110]. Additionally, retroviral vectors may present a health hazard if applied to human gene therapy, because the introduction of retrovirus DNA sequences with their potent regulatory elements can lead to the undesired, hazardous expression of non-globin genes in erythroid cells.
Use of adeno-associated vectors for the introduction of a human .gamma.-globin gene into erythroid cells has also been performed [Walsh, C. E., et al., (1992) Proc. Natl. Acad. Sci. USA 89:7257-7261] with successful expression of the .gamma.-globin gene. However, it was recently found that adeno-associated virus (AAV) interferes with normal cellular regulation [Winocour, E., et al., Virology (1992) 190:316-329].
In contrast, the present invention provides for the first time, an SV40-derived vector encoding, for example .beta.-globin, that can be effectively introduced into human erythroid cells, for example erythroid cells from .beta.-thalassemia patients, and be expressed therein at high levels to provide approximately normal levels of .beta.-globin, thereby providing a way for the gene therapy treatment of .beta.-thalassemia. The SV40-derived vectors have a number of advantages over the adeno-derived vectors or the retrovirus-derived vectors, the major ones being the safety of using SV40 for human administration, the ease of manipulation of SV40 vectors and the possibility to prepare SV40 pseudovirions for infection of erythroid cells.
The constructs of the invention may also be useful in delivering P-glycoprotein, encoded by the human MDR1 gene, in bone marrow autografting. As will be shown, the SV40 pseudoviral vector is most promising for ex vivo (and probably also in vivo) gene therapy via the bone marrow (BM), most probably an excellent vector for the treatment of bone marrow autografts. Such autografting procedures with MDR1 transfer are already approved for use in the United States.
The autografting of hemopoietic stem cells from bone marrow or peripheral blood to rescue patients from high dose chemotherapy has been explored intensively in lymphomas, Hodgkin's Disease, and solid tumors [Armitage J. and Gale R (1989) Am J Med 86:203-209; Frei E., et al., (1989) J Clin Oncol 7:515]. The major limitation of conventional bone marrow autografting is that the patient is still left for a period of several weeks without a functional bone marrow during the chemotherapy-induced nadir at which time, mortality and morbidity are high. A promising approach to avert this problem is the conferring of chemotherapy resistance to the patients' own BM cells prior to treatment. These cells then repopulate the BM and allow the administration of more intense, curative chemotherapy. An additional benefit anticipated from chemotherapy-resistant autografting (besides lowering patient mortality) is the need for fewer days in hospital and less need for antibiotics. This would greatly lower the overall cost of the autotransplantation procedure, which is is an important consideration in these days of concern over the rising costs of health care.
A major hinderance to performing gene therapy is the lack of an efficient procedure for introducing cloned DNA into primary human cells. Gene transfer using viral infection, in particular using retroviral vectors, has been widely applied as a solution to this problem. Retroviral vectors are efficient vectors for murine cells. However, as already mentioned above, many problems have been encountered in their use with human cells, in particular with stem cells. These will be discussed in detail below. In addition, there are well-founded fears regarding the use of retroviral vectors as to their potential for inducing malignancies in recipient cells. This concern is based on the fact that the natural sources of these viruses are various animal tumors.
The MDR1 gene [Pastan I. and Gottesman M. (1991) Ann Rev Med 42:277-286] encodes a 170 kd plasma transmembrane glycoprotein (P-glycoprotein), which confers energy-dependent resistance to a number of naturally-occurring, structurally unrelated types of chemotherapeutic agents, including anthra-cyclines, vinca alkaloids, epipodophyllotoxins, taxol, and actinomycin D [Pastan and Gottesman (1991) ibid.]. Although this gene was first identified in tumor cells which overexpresssed P-glycoprotein (Fojo A., et al., (1985) Proc Natl Acad Sci USA 82:7661-7665], the MDR1 gene product is expressed in normal body tissues, at variable levels. These range from very high levels in kidney tubule and colonic epithelium cells to very low levels in most human peripheral blood white blood cells [Fojo, A., et al. (1987) Proc Natl Acad Sci USA 84:265-269; Klimecki W. T., et al., (1994) Blood 83:2451-2458].
It was recently recognized that a small population of normal human BM cells also express MDR1, which are probably stem cells [Chaudhary P, Roninson I (1991) Cell 66:85-94]. A subset of normal peripheral blood lymphocytes which express the CD56 surface antigen have also been found to express high levels of MDR1 (Klimecki et al. (1994). ibid.] However, the overall expression and/or the total number of MDR1-expressing cells in normal human bone marrow and peripheral blood is necessarily low, since this tissue is so highly chemosensitive [Noonan K, et al., (1990) Proc Natl Acad Sci USA 87:7160-7164]. Recent evidence has shown a lack of functional P-glycoprotein in normal granulocytes [Klimecki et al. (1994) ibid.]. This forms the basis for MDR1 gene transfer experiments.
Recent experiments in tissue culture cells showed that a cDNA for the human MDR1 gene conferred resistance to cytotoxic agents [Ueda K, et al., (1987) Proc Natl Acad Sci USA 84:3004-3008; delaFlor-Weiss E, et al., (1992) Blood 80: 3106-3111]. Transgenic mice expressing a human MDR1 gene showed long-term resistance to chemotherapy-induced neutropenia [Mikisch G., et al. (1992) Blood 79:1087-1093]. In another work, murine BM cells expressing human MDR1 were also resistant to chemotherapy-induced neutropenia [Sorrentino B, et al., (1992) Science 257:99-103]. In a further work, live mice transduced with an MDR1 gene showed expression of the gene in granulocytes and in bone marrow cells [Podda S., et al., (1992) Proc Natl Acad Sci USA 89:9676-9680]. All these experiments were performed using retroviral vectors. New retroviral vectors have been developed [delaFlor-Weiss, E. et al. (1992) ibid.; Ward, M., et al. (1992) Blood 80:(Suppl):239a] but as yet, low titers and low transduction efficiency are still a problem, as demonstrated in human tissue culture cells [delaFlor-Weiss et al. (1992) ibid.; Podda et al. (1992) ibid.].
Additional recent experiments have been reported using retroviral vectors [Hanania, E. et al. (1993) Blood 82(Suppl):216a; Sorrentino B, et al. (1993) Blood 82(Suppl):216a; Ward et al. (1993) ibid.]. In these experiments, the retroviral infection was complicated and difficult to perform. Retroviral vectors require dividing cells in which to integrate and the human BM stem cell is not usually in cycle. Therefore, pretreatment of the cells with multiple growth factors (IL6, IL3, and stem cell factor) was required [Ward M, et al. (1994) Blood 84:1408-1414]. This dictated that the number of cells to be infected be reduced to a minimum, thus necessitating the use of columns for separation of CD34 positive cells (Ward et al. (1993) ibid.; Ward et al, (1994) ibid.], which are the putative stem cells in the bone marrow. The requirement for CD34 separation and use of multiple growth factors makes the procedure highly labor intensive and exceedingly costly. Each column for CD34 separation costs many thousands of dollars, as do the cytokines required for stem cell collection and stimulation to allow for retroviral integraton. Some of these cytokines, such as stem cell factor are at present only available in very limited amounts, precluding their therapeutic use in patients. The high cost of the procedure is an important consideration and a major hinderance in planning the broad scale application of such treatments.
These experiments further demonstrated a number of the other problems which are inherent in the use of retroviral vectors. Murine stem cells are usually transduced at low efficiency, and primate stem cells at even lower efficiency [Sorrentino et al. (1992) ibid.]. Using retroviral vectors in human CD34+ cells, a low transfection efficiency (5-9%) was demonstrated [Ward et al. (1993) ibid.]. In murine cells and in long term bone marrow cultures (Hanania et al. (1993) ibid.; Sorrentino et al. (1993) ibid.], LTR elements were required as promoters to provide expression at high levels. Although infectivity of cells was documented, it was generally by use of PCR technique, which will be positive even if only a small number of cells is infected. Furthermore, the group of researchers who performed the most intense RNA analysis discovered that aberrant splicing occured, deleting much of the coding sequence of the MDR1 protein [Sorrentino et al. (1993) ibid.]. This resulted in truncated mRNA products, which reduced the expression of the transfected gene [Sorrentino et al. (1993) ibid.]. This is typical of many previous attempts at using retroviral vectors for gene therapy of other diseases. These vectors are not faithful in their ability to transmit an exogenous gene, and rearrangement or deletion of the gene of interest is a frequent event.
These findings suggest that development of alternative vectors is desirable and the SV40 pseudoviral vector is potentially more suitable for the purpose of MDR1 expression in bone marrow cells.
The constructs of the present invention may also be used in the treatment of APO A-I associated atherosclerosis. Lipid transfer in the circulation is performed via lipoprotein particles which are composed of apoproteins, triglycerides, phospholipids, cholesteryl ester and free cholesterol. The lipoprotein particles are separated by-density, determined by the lipid/protein proportion in the different particles. The lower density particles (LDL, VLDL, and remnant APO B-containing particles) transfer cholesterol and triglycerides from the liver and the intestine to the peripheral tissues. High levels of these particles contribute to the development of atherosclerosis, the leading cause of heart disease.
Results from epidemiological studies indicate a reverse correlation between High Density Lipoprotein (HDL) levels and susceptibility to atherosclerosis [Gordon, D. J., et al. N Engl J Med (1989) 321:1311-1316]. The importance of HDL levels in the development of atherosclerosis has been demonstrated in human and also in animal models: Trials using lipid lowering drugs revealed that an increase in HDL cholesterol was associated with decreased incidence or progression of coronary heart disease (CHD). Families with inherited hyperalphalipoproteinemia syndrome (high HDL concentrations) tend to be protected from CHD, and families with hypoalphalipoproteinemia (low HDL) show high prevalence of CHD. In experiments with animal models cholesterol accumulation in the developing atherosclerotic lesions is affected by HDL levels. A recent study done with transgenic mice overexpressing human APO -AI gene demonstrates a positive correlation between APO -AI levels and HDL cholesterol. The high level of HDL obtained in these mice reduces the rate of development of fatty streaks in the aorta under atherogenic diet [Rubin, E. M., et al. Nature (1991) 353:265-267].
Furthermore, breeding APO E deficient mice which were severely hypercholesterolemic and developed advanced atheroma independent of dietary cholesterol, with human APO A-I transgenic mice did not affect the elevation in plasma cholesterol but an increase in HDL was observed, associated with six-fold decrease in atherosclerosis [Paszty, C., et al. J Clin Invest (1994) 946:899-903; Plump, A. S., et al. Proc Natl Acad Sci USA (1994) 91:9607-9611].
Genetic defects in the synthesis of APO A-I result in very low HDL levels and premature atherosclerosis [Breslow, J. L. J Clin Invest (1989) 84:373-386]. 9% of the patients with premature coronary artery disease (CAD) suffer from hypoalphalipoproteinemia [Schaefer, E. J., et al. Elsevier (1986) 11:79-86]. This familial disorder is characterized by very low HDL-C level, while the level of the other lipoprotein particles remains normal.
Drugs and factors that usually raise HDL-C levels (exercise conditioning, alcohol intake, estrogens and drugs like nicotinic acid and fibrates) proved to be ineffective in these patients, who are at increased risk for early death as a result of heart disease.
As APO A-I levels determine the HDL-C levels, gene therapy with normal APO A-I promises a new therapeutic approach to this problem.
Other advantages of SV40-derived vectors are set forth herein below in detail.